Study On The Effects Of Sites Within The Receptor-binding Domain Of H9N2 Subtype Influenza Virus On Its Receptor Affinity

Although H9N2 subtype is a low pathogenicity influenza virus,it has a wide host range,which not only infects poultry,but also breaks through the interspecies barrier to infect humans and other mammals.Therefore,it is important for epidemiology and public health significance to study the molecular mechanism of the potential of infection and cross-species transmission of H9N2 subtype influenza virus.In the life cycle of influenza virus,it is the first step for virus internalization and invasion into host cells of the binding between the virus hemagglutinin(HA)and the sialic acid receptors on host cells.According to the sequence analysis of the 220-loop(positions 221~228)of the HA receptor-binding domain of H9N2 subtype influenza viruses available in Gen Bank,it was found that the amino acids at positions 226 and 227 are only relatively conservative.Among them,the amino acid at position 226 is mainly L,Q,or M,while that at position 227 is Q or M.Therefore,it is necessary to explore the effects of different amino acids at these two positions on the viral receptor affinity.In this study,the amino acids at positions 226 and 227 in the HA of A/Chicken/Jilin/DH102/2012(H9N2)in the Y280-like sublineage were substituted by the overlapping PCR,and then the site-directed mutants Re DH102/L226 Q,Re DH102/L226 M,and Re DH102/Q227 M,as well as the non-mutant Re DH102,were rescued by the reverse genetics technique.The results of solid-phase enzyme receptor-binding assay showed that L-226-containing virus prefers binding to human-like receptors;Q-226-containing virus binds to avian-like receptors in priority;M-226-containing virus can bind to both two types of receptors,despite of preferentially binding to human-like receptors.For those viruses possessing Q or M at position 227,they prefer human-like receptors.Therefore,compared with the substitution at position 227,the Q226 L mutation is enough to alter the receptor preference of virus.It is speculated that 226 L is an important determinant site of H9N2 virus infection in mammals,given that the amino acid at position 226 of HA is L for more than 90% of H9N2 virus isolates in recent years.The results of the tissue-binding capacity showed that four viruses can effectively bind to three sources of lung tissues from SPF chicken,SPF BALB/c mouse,and human with no significant difference within group and between groups,indicating that they have the potential of cross-species transmission.The results of indirect immunofluorescence assay and flow cytometry demonstrated that the affinity of Re DH102/L226 Q and Re DH102/Q227 M for CEF and A549 cells expressing the avian-like receptors was significantly higher than that of Re DH102(p<0.0001).And the affinity of Re DH102/L226 M for A549 cells was higher than that of Re DH102(p<0.05).The difference of binding capacity of these four strains for these two kinds of cells indirectly reflected their receptor affinity,which confirmed the results of solid-phase enzyme-linked receptor-binding assay.However,there was no significant difference between the binding capacity of these four viruses for MDCK cells due to an almost equivalent amount of avian-like and human-like receptors on the surface of MDCK cells.All these indicate that the four viruses have the potential cross-species transmission at the cellular level because they were able to bind well to three sources of cells.It was significantly different for them to multiply on MDCK cells,although there was no obvious difference in the binding capacity of the four viruses for MDCK cells.The L226 Q mutation decreased the multiplication capacity of virus(p<0.01),showing a decrease in the highest titer and a significantly smaller plaque size.There was no obvious effect on the multiplication ability and plaque size of the L226 M mutated virus.The Q227 M mutation can slightly lower the multiplication ability,but had no effect on the plaque size.All these results suggested that the site-directed mutations at positions 226 and 227 within the HA receptor-binding domain influence the receptor affinity of H9N2 virus and their binding capacity for different sources of cells to different degree.Compared with the position 227,the mutations at position 226,especially the substitution between Q and L,will significantly change the receptor affinity,cell tropism and the proliferation on MDCK cells of virus.Therefore,it is an important amino acid site of the position 226 at the HA receptor-binding domain to determine the potential of cross-species transmission of H9N2 subtype influenza virus.